Oral formulations of pyrrolidine derivatives

ABSTRACT

The present invention relates to solid oral formulations comprising a compound of formula (3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime, and/or an active metabolite thereof, and the use of said formulations in the treatment and/or prevention of preterm labor, premature birth, dysmenorrhea and embryo implantation failure due to uterine contractions. The present invention is furthermore related to processes for their preparation.

TECHNICAL FIELD

The present invention relates to solid oral formulations comprising acompound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, and the use of said formulations inthe treatment and/or prevention of preterm labor, premature birth,dysmenorrhea and embryo implantation failure due to uterinecontractions. The present invention is furthermore related to processesfor their preparation.

BACKGROUND OF THE INVENTION

Oxytocin (OT) is a cyclic nona-peptide that mediates its physiologicalactions through activation of the oxytocin receptor (OT-R), a cellmembrane receptor belonging to the class of G protein-coupled receptorsthat is similar to arginine vasopressin receptors. One important actionof OT is to cause the contraction of the uterus of mammals during labor.Repeated, concerted and regular contraction of the uterus will cause thedilatation of the cervix, the rupture of foetal membranes and lead toexpulsion of the foetus. Premature labor is when these contractionsoccur before the normal term of pregnancy. Preterm increase of uterineactivity is the most common expression of preterm labor.

Premature labor leads to undesired premature birth, a serious healthproblem that remains the major cause of perinatal mortality and severemorbidity, especially respiratory distress syndrome, intraventricularhaemorrhage, bronchopulmonary dysplasia and necrotising enterocolitisthat are far more common in preterm than in term infants. Long-termimpairments such as cerebral palsy, visual impairment and hearing lossare also more common in preterm infants. Nowadays, preterm birth remainsthe leading cause of infant mortality and morbidity in industrializednations, where, despite the significant improvements in obstetricalmedicine, it is causing high costs for neonatal intensive care ofpremature babies. The actual costs are even higher to society whentaking into consideration the healthcare provision of pretermchildbirth-related ailments, such as respiratory distress syndrome,heart conditions, cerebral palsy, epilepsy, and severe learningdisabilities. The management of preterm labor represents a significantproblem in the field of obstetrics.

The OT/OT-R system plays a vital role in initiating labor in mammals, inparticular in humans. The density of OT-R increases markedly in themyometrium before the onset and during labor. Also it is thought thatthe local OT peptide hormone concentration increases markedly beforeparturition in human. The high circulating concentrations ofprogesterone induce uterine quiescence while the uterus acquirescontractile ability. Shortly before term, plasma progesteroneconcentrations fall, OT-R expression in the uterus increases markedly,OT is released and uterine contractile activity increases. At term, thecontractions rise to a crescendo, resulting in delivery as a result oftwo interacting positive feedback loops. The first is a local uterineloop: within the uterus itself, contractile prostaglandins are producedand released in response to OT and uterine contractions. Theseprostaglandins may play a further role in cervical ripening andweakening of fetal membranes. The second loop involves the hypothalamus:in response to uterine contractions and vaginal and cervical distension,magnocellular oxytocin neurons in the hypothalamus increase theiractivity resulting in the release of OT from their axon terminals in theposterior pituitary. The released OT acts upon the uterus both tostimulate the further production of prostaglandins and to contributefurther to the contractions of the uterus.

Therefore, blocking the effect of OT by antagonizing OT-R mightrepresent an attractive modality for the treatment of diseases relatedto the OT-R activity, in particular preterm labor, premature birth anddysmenorrhea.

Tocolytic, i.e. uterus relaxing agents, have been used in clinicalstudies for the pharmaceutical treatment of preterm labor. Most of theseagents are used off-label. They have shown very limited efficacy, ifany, in prolonging gestation and without clear demonstration ofimprovement of neonate outcome. Current tocolytics are very oftenassociated with unwanted adverse effects on women, foetus or neonate.Such tocolytics include beta-2-adrenergic agonists, prostaglandinsynthesis inhibitors, magnesium sulfate, nitric acid donors and calciumchannel blockers. Beta-2-adrenergic agonists such as ritodrine orterbutaline cause a number of cardiovascular and metabolic side effectsincluding maternal tachycardia, palpitations, hypotension, alteredthyroid function and fetal and neonatal hypoglycaemia, tachycardia.Ritodrine is no longer FDA approved. The calcium channel blockernifedipine is also a medicine that is used to try to stop contractions.Some of the side effects that may occur include facial flushing,headache, nausea, palpitations, and lightheadedness. The totalprostaglandin synthesis inhibitor (NSAID) indomethacin has been used. Itcan also have serious effects on the fetus: constriction of ductusarteriosus, pulmonary hypertension, decrease in renal function witholigohydramnios, intraventricular hemorrhage, hyperbilirubinemia,necrotizing enterocolitis. Maternal side effects include abdominaldiscomfort, nausea, vomiting, depression and dizzy spells for themother. Another NSAID is sulindac that has a side effect profile similarto indomethacin. For magnesium sulfate, meta-analyses have failed tosupport it as a tocolytic agent. Women reported important side effectssuch as flushing, lethargy, headache, muscle weakness, pulmonary edemaand cardiac arrest. A newborn that has been exposed to magnesium sulfatemay show lethargy, hypotonia, respiratory depression, bone problems,osteopenia and fractures. Recently, the FDA is advising healthcareprofessionals against using magnesium sulfate injection for longer than5-7 days to stop preterm labor in women.

Atosiban, a dual vasopressin V1a receptor and OT-R antagonist ismarketed in EU and used to stop contractions and delay preterm deliveryby a few days. The principal drawback to the use of peptide antagonistslike Atosiban is the problem of low oral bioavailability resulting fromintestinal degradation. Hence, they must be administered parenterally.

The development of orally active small molecule antagonists that areselective for the OT-R is expected to overcome these problems.Pyrrolidine derivatives being OT-R antagonists are disclosed in WO01/72705, WO 02/102799, WO 2002/074741, and WO 2004/005249.

Thus, in the management of preterm labor and premature birth, there is aneed for an oral formulation which is convenient to administer, which issuitable for providing a fast onset of action and which provides a goodbioavailability of a compound being an OT-R antagonist.

SUMMARY OF THE INVENTION

The present invention provides a dispersible tablet comprising acompound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and at least one or more pharmaceutically acceptable excipients.

The invention also provides said dispersible tablet, for use in thetreatment and/or prevention of disorders selected from the groupcomprising preterm labor, premature birth, embryo implantation failuredue to uterine contractions, dysmenorrhea, premature ejaculation, sexualdysfunction, endometriosis, infertility, benign prostatic hyperplasia,neuro-psychiatric disorders, autism, social behavior disorders,psycho-social stress, and/or cardiovascular disorders.

Also provided is a process for the preparation of said dispersibletablet characterized by a step of wet granulation.

The invention further provides a kit comprising said dispersible tablet,and information for use thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A and FIG. 1B: Plasma concentration profiles of solid oralformulations in the dog. FIG. 1A shows the plasma concentration (ng/ml)vs. time profile of formulations 1 (10% granules), 2 (5.8% granules), 3(dispersible tablets) and 4 (conventional tablets) over the time periodfrom 0 to 72 h. FIG. 1B shows an enlargement of FIG. 1A for the periodfrom 0 to 12 h.

FIG. 2: Individual plasma concentration profiles of formulation 3(dispersible tablets) in the dog. Plasma concentration of the compoundof formula (3Z,5S) is measured (ng/ml) for each dog (n=5) for the timeperiod from 0 to 12 h.

FIG. 3: Individual plasma concentration profiles of formulation 4(conventional tablets) in the dog. Plasma concentration of the compoundof formula (3Z,5S) is measured (ng/ml) for each dog (n=5) for the timeperiod from 0 to 12 h.

FIG. 4: Schematic TLC profile of the collected fractions from dry flashchromatography of crude(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3oneO-methyloxime. Fraction 1-5: eluted with pure toluene. Fractions 6-10:eluted with toluene/MeOH 1% vol/vol. Fractions 10 to 15: eluted withtoluene/meOH 2% vol/vol.

DETAILED DESCRIPTION OF THE INVENTION

Generally, the present invention relates to a solid oral formulationcomprising a compound of formula5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,its geometrical isomers, its optically active forms as enantiomers,diastereoisomers, mixtures of these, its racemate forms as well asactive metabolites thereof, and at least one or more pharmaceuticallyacceptable excipients.

Preferably, the present invention relates to a solid oral formulationcomprising a compound of formula(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, and at least one or morepharmaceutically acceptable excipients.

Even more preferably, the present invention relates to a solid oralformulation comprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, and at least one or morepharmaceutically acceptable excipients.

The compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximealso named (3Z,5S) herein is produced by methods such as those disclosedfor example in WO2004/005249 and WO2005/082848.

Usually, said compound is synthesized and obtained in isomeric mixtures(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximecomprising(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeand(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime.

In case the isomer Z is preferred, then the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximesynthesized and obtained in isomeric mixtures is purified according tomethods disclosed in PCT/EP2014/066075.

Thus, the purity of the compound(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximein said isomeric mixtures is at least 85% to 100%, preferably 85% to99.9%, more preferably 90% to 99.9%, and even more preferably 95% to99.9%.

Alternatively, the present invention relates to a solid oral formulationcomprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, provided in substantially pureform, and at least one or more pharmaceutically acceptable excipients.

As used herein, the term “substantially pure” refers to a compoundprovided in a form which is substantially free of other compounds.Examples of said “other compounds” include(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one,(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneoxime,(3R,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-3-methoxyamino-pyrrolidine,(3S,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-3-methoxyamino-pyrrolidine,(3Z,5S)-5-(O-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeand(3E,5S)-5-(O-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime.

Most preferably, the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof is substantially free of thecompound of formula(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime.

Even more preferably, the purity of a substantially pure form compoundof formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, is at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, at least 99%, at least 99.2%, atleast 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least99.7%, at least 99.8%, at least 99.9% or at least 100% and is thereforesubstantially free of compound of formula(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime.

Even more preferably, the purity of the substantially pure form compoundof formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, is at least in the range of 85% to100%, preferably 85% to 99.9%, more preferably 90% to 99.9%, and evenmore preferably in the range of 95% to 99.9%.

As used herein, the term “active metabolite thereof” refers to a productproduced through metabolism in the body, or in vitro, of a specifiedcompound, i.e. in the present case(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeand which exhibits the same biological activity as(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime.

Active metabolites of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximemay be identified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchmetabolites may result for example from the oxidation, glucuronidationor other conjugation, hydrolysis, reduction and the like, of theadministered Z form. Accordingly, the invention includes activemetabolites of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,including compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof. Such metabolite may also beproduced in vitro by oxidation, reduction, hydrolysis, glucuronidationor other conjugation transformation of the corresponding(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime.Examples of actives metabolites of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,include compounds whose structures are shown below:

A compound which, upon administration to the recipient, is capable ofbeing converted into a compound of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof as described above, is known as a“prodrug”. A prodrug may, for example, be converted within the body, e.g. by hydrolysis in the blood, into its active form that has medicaleffects. Pharmaceutical acceptable prodrugs are described in T. Higuchiand V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A. C.S. Symposium Series (1976); “Design of Prodrugs” ed. H. Bundgaard,Elsevier, 1985; and in Edward B. Roche, ed., Bioreversible Carriers inDrug Design, American Pharmaceutical Association and Pergamon Press,1987, which disclosures are incorporated herein by reference.

As used herein, the term “solid oral formulation” refers to a tablet, adispersible tablet, a fast dissolving tablet, a quick dissolving tablet,a fast melt tablet, a mouth-dissolving tablet, a melt-in mouth tablet,an orodispersible tablet, a lyophilised unit, a porous tablet, aconventional tablet, a coated tablet, an uncoated tablet, agastro-resistant tablet, an effervescent tablet, a soluble tablet, achewable tablet, an oral lyophilisate, a powder, an oral powder, apellet, a capsule and/or a granule. Preferably, the solid oralformulation is a tablet, more preferably, a dispersible tablet.

As used herein, the term “dispersible tablet” includes a disintegratingtablet that is swallowed, or intended to be disintegrated rapidly inwater and to be swallowed.

As used herein, “pharmaceutically acceptable excipients” includes anycarriers, diluents, adjuvants, vehicles, preserving agents, antioxidantagents, fillers, bulking agent, glidant, buffering agents, thickeningagents, disintegrating agents, lubricants, binders, wetting agents,sweeteners, flavouring agent, taste-masking agents, emulsifying agents,suspending agents, solvents, dispersion media, coatings, antibacterialagents, anti-oxidants, antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutical active substances is well-known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the solid oral formulation iscontemplated. Supplementary active ingredients can also be incorporatedinto the solid oral formulation as suitable therapeutic combinations.

Preferably, the dispersible tablet of the invention comprises a compoundof formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeand at least one or more pharmaceutically acceptable excipients selectedfrom the group consisting a disintegrant, a wetting agent, a carrier, alubricant, a binder, a diluent, a sweetener, and a taste-masking agent.

Thus, the present invention relates to a dispersible tablet comprising acompound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and a disintegrant. For example, the “disintegrant” may be selected fromone of the group consisting of sodium croscarmellose, crospovidone,sodium alginate, colloidal magnesium-aluminum silicate, calciumsilicate, sodium starch glycolate, acrylic acid derivatives,microcrystalline cellulose, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, modified cellulose gum, cross-linked povidone,alginic acid and alginates, pregelatinised starch, modified corn starchand combinations thereof. Preferably, the “disintegrant” is selectedfrom the group consisting of sodium croscarmellose, crospovidone andcombination thereof. More preferably, the “disintegrant” is sodiumcroscarmellose.

The present invention alternatively relates to a dispersible tabletcomprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and a wetting agent. For example, the “wetting agent” is selected fromthe group consisting of poloxamer, sodium lauryl sulphate,polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearate,sorbitan fatty acid esters and combinations thereof. Preferably, the“wetting agent” is selected from the group consisting of poloxamer,sodium lauryl sulfate and combination thereof. More preferably, the“wetting agent” is poloxamer 188.

Furthermore, the present invention relates to a dispersible tabletcomprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and a carrier. For example, the “carrier” may be selected from the groupconsisting of calcium silicate, calcium carbonate, calcium phosphate,tribasic calcium phosphate, lactose, starch, modified starch, sugars,celluloses, cellulose derivatives, polymethacrylates, chitin, chitosanand combination thereof. Preferably, the “carrier” is selected from thegroup consisting of calcium silicate, calcium carbonate, calciumphosphate and combinations thereof. More preferably, the carrier iscalcium silicate.

The present invention also relates to a dispersible tablet comprising acompound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or a disintegrant, and/or a wetting agent, and/or a carrier.Preferably, said dispersible tablet comprises a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or sodium croscarmellose, and/or poloxamer 188, and/or calciumsilicate.

The present invention also relates to a dispersible tablet comprising acompound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeand at least one or more pharmaceutically acceptable excipients in anamount effective to provide a tablet that releases between about 90 to100% of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime.A classical Pharmacopeia compliant dissolution test (USP2) wasperformed. As shown in the example, a rapid dissolution profile of thedispersible tablet of 200 mg is observed at 15 min wherein theconcentration of (3Z,5S) in water is between 90% to 100% of the initialconcentration value (Table 30).

Preferably the one or more pharmaceutical acceptable excipients includesat least one disintegrant. More preferably, the disintegrant is selectedfrom the group consisting of sodium croscarmellose, crospovidone and acombination thereof. More preferably, the “disintegrant” is sodiumcroscarmellose.

For example, the “binder” may be selected from the group consisting ofpolyvinylpyrrolidone, cross-linked PVP, cellulose or cellulosederivatives such as hydroxypropylmethyl cellulose (HPMC),carboxymethylcellulose sodium, ethyl cellulose, hydroxyethyl cellulose,hydroxypropyl cellulose, methyl cellulose, carboxyethylcellulose,calcium, guar gum, tragacanth, polyvinylacetates, gelatin,pregelatinised starch, starch, polyvinylalcohols, alginic acid, sodiumalginate, sorbitol, glucose, magnesium aluminium silicate, dextrin,polyethylene glycol, polymethacrylates and combination thereof.

For example, the “diluent” may be selected from the group consisting ofmicrocrystalline cellulose, lactose monohydrate, lactose, compressiblesugar, sugar, dextrose, mannitol, dextrin, maltodextrin, sorbitol,xylitol, sodium chloride, calcium carbonate, magnesium carbonate,calcium phosphate, calcium sulphate, magnesium oxide, kaolin, powderedcellulose, pregelatinized starch, starch, barium sulphate, magnesiumtrisilicate, aluminium hydroxide and combinations thereof.

For example, the “sweetener” may be sodium saccharine, sucrose,sucralose, aspartame, sorbitol or combination thereof.

For example, the “lubricant” may be selected from the group consistingof glycerol dibehenate, glycerol tribehenate, magnesium stearate,calcium stearate, talc, sodium stearyl fumarate, sodium behenate,stearic acid, cethyl alcohol, polyoxyethylene glycol, leucine, sodiumbenzoate, stearates, talc, polyethylene glycol, glyceryl monostearate,glyceryl palmitostearate, liquid paraffin, poloxamer, sodium laurylsulphate, magnesium lauryl sulphate, hydrogenated castor oil, colloidalsilicon dioxide, palmitostearate, stearic acid, zinc stearate, stearylalcohol, hydrogenated vegetable oil and combinations thereof.

The present invention also relates to a solid oral formulationcomprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, and at least one or morepharmaceutically acceptable excipients, wherein the concentration ofsaid compound and/or active metabolite thereof, is comprised betweenabout 1% and 50% w/w.

Preferably, the concentration of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof is 10-40% w/w, 20-30% w/w, about 20%w/w.

As used herein, the term “about” applies to numeric values and refers toa range of numbers that one of skill in the art would considerequivalent to the recited values. For example, “about 20% w/w” refers tothe range 15%-25% w/w.

The present invention also relates to a solid oral formulationcomprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, and at least one or morepharmaceutically acceptable excipients, wherein said formulationcomprises about 10 mg to about 500 mg of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof. Preferably, said formulationcomprises about 20-400 mg or 40-200 mg of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof. Preferably, said formulation in theform of a dispersible tablet comprises about 50 mg or 200 mg of acompound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime.

Advantageously, the present invention provides a solid oral formulation,which is i) convenient to administer, ii) suitable for providing a fastonset of action and which provides a good bioavailability of thecompound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or active metabolite thereof. As used herein, the term “Tmax” refersto the time to reach the peak plasma concentration (Cmax) of a drugafter administration wherein the concentration is the amount of the drugin a given volume of plasma, expressed in ng/ml in the examples.

As used herein, the term “onset of action” refers to the time requiredafter administration of a drug to become effective.

As shown in the examples, the solid oral formulations of the presentinvention, in particular in the form of a dispersible tablet, have theadvantage of being suitable for providing a rapid onset of action. Themaximum concentration in blood of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or active metabolite thereof, is reached at a time Tmax less than 5h, preferably less than 4 h, more preferably less than 3 h, less than 2h, less than 1.5 h, even more preferably less than 1 h followingadministration of said solid oral formulation.

Also, the maximum concentration in blood of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or active metabolite thereof, is reached at a time Tmax between 0.5to 4 hours, 0.5 to 2 hours, preferably 0.5 to 1.5 hours, more preferablyat a time between 0.5 to 1 hour following administration of said solidoral formulation. Preferably, said solid oral formulation is adispersible tablet.

Noteworthy, at a time of 0.5 hour following administration of the solidoral formulation, the concentration in blood of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or active metabolite thereof, is at least 25%, at least 35%, atleast 40%, at least 45%, at least 55%, at least 65%, at least 75%, or atleast 85% of Cmax. Preferably, said solid oral formulation is adispersible tablet.

Also, at a time of 0.5 hour following administration of the solid oralformulation, the concentration in blood of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or active metabolite thereof, is comprised between 35% to 100%, 45%to 100%, 55% to 100% of Cmax, preferentially 57% to 92% of Cmaxindicating that the solid oral formulation is suitable for providing arapid onset of action. Preferably, said solid oral formulation is adispersible tablet.

As shown in the examples, at a time of 0.5 hour following administrationof the dispersible tablet, the concentration in blood of the compound offormula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,is comprised between 59% to 100% of Cmax in animal (Table 17),preferentially 57% to 92% of Cmax in human subjects (Table 22).

Thus, the present invention provides a solid oral formulation,preferably a dispersible tablet that is suitable for providing a rapidonset of action, which is crucial for the management of preterm laborand premature birth.

In particular, it has been shown that the maximum concentration of theactive substance (3Z,5S) is detected rapidly at about 4 hours, 2 hours,1.5 hours, or 1 hour following administration of the dispersible tabletof the present invention.

Furthermore, the solid oral formulation of the present invention ischaracterized by a bioavailability of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or active metabolite thereof, comprised between 50-100%, and/or50-99%. Preferably, said bioavailability is comprised between 75-100%,or 75-99%, more preferably, between 80-100%, or 80-99%.

As used herein, the term “bioavailability” (F %) refers to the fractionof an administered dose of a product that reaches the systemiccirculation. By definition, when the product is administeredintravenously, its bioavailability is 100%. When the product isadministered via other routes, its bioavailability generally decreases.

As shown in the examples, the bioavailability (F %) of the solid oralformulations of the present invention, is comprised between 58% and 90%(Table 16). In particular, the bioavailability of the solid oralformulation in the form of a dispersible tablet is comprised between 80%and 100% (Table 18, 102% in table 18 is indicated as 100% based on thestandard deviation).

In particular, it has been shown that the bioavailability of the activesubstance (3Z,5S) is about 89% following administration of thedispersible tablet of the present invention (Table 16).

Advantageously, patients administered with the solid oral formulation ofthe present invention will benefit from a fast onset of action and/or agood bioavailability.

Also, the present invention provides a solid oral formulationcomprising: 20% by weight of a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof;

1-20% by weight of calcium silicate;

0.1-20% by weight of PVP30K;

0.01-5% by weight of poloxamer 188;

0.5-20% by weight of sodium croscarmellose;

1-90% by weight of microcrystalline cellulose 112;

1-90% by weight of lactose monohydrate;

0.01-0.5% by weight of sodium saccharine; and

0.1-10% by weight of glycerol dibehenate.

Alternatively, the present invention provides a dispersible tabletcomprising 20% by weight of a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and 0.5-20% by weight of a disintegrant. Preferably said disintegrant issodium croscarmellose.

Also alternatively provided is a dispersible tablet comprising 20% byweight of a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and 1-20% by weight of a carrier. Preferably, said carrier is calciumsilicate.

Alternatively, it further provides a dispersible tablet comprising 20%by weight of a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and 0.01-5% by weight of a wetting agent. Preferably, said wetting agentis poloxamer 188.

Preferably, said solid oral formulation consists of:

20% by weight of a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof,

5% by weight of calcium silicate,

1% by weight of PVP30K,

2% by weight of Poloxamer 188,

5% by weight of Sodium croscarmellose,

15% by weight of Microcrystalline cellulose 112,

47.8% by weight of Lactose monohydrate,

0.2% by weight of Sodium saccharine and

4% by weight of Glycerol dibehenate.

Whilst the compound(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeand/or the active metabolite thereof may be used as the sole activeingredient of the solid oral formulation, it is also possible for thecompound to be used in combination with at least one or more furtheractive compounds. Such further active compounds may be further compoundsaccording to the invention, or other active compounds selected from thegroup comprising calcium channel blockers, magnesium sulfate, selectiveprostaglandin modulators, beta-2-adrenergic agonists, beta-3-adrenergicreceptor agonists, and/or corticosteroids.

Preferably, corticosteroids are selected from the group comprisingBetamethasone and Dexamethasone, and/or salts thereof. Thesecorticosteroids are given before birth to accelerate a preterm fetus'lung development and maturation to prevent respiratory distress syndrome(RDS) and other related complications following premature birth.

Alternatively, the solid oral formulation of the invention can beadministered concomitantly or separately with at least one compoundselected from the group comprising calcium channel blockers (such asnifedipine), magnesium sulfate, prostaglandin receptors modulators (suchas agonists or antagonists of either EP1 or EP2 or EP3 or EP4 or FPreceptors), prostaglandin synthesis inhibitors (such as indomethacin,nimesulide, sulindac, rofecoxib, celecoxib), beta-2-adrenergic agonists(such as ritodrine, terbutaline, salbutamol), beta-3-adrenergic receptoragonists, nitric acid donors (such as glyceryl trinitrate) and/orcorticosteroids (such as dexamethasone, betamethasone).

As used herein, the term “concomitantly” refers to the administration ofa solid oral formulation comprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, which is then immediately followedby the administration of at least one compound selected from the groupdisclosed supra.

As used herein, the term “separately” encompasses sequential orsubsequent administration and refers to the administration of a solidoral formulation of the invention comprising the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, followed by a time period ofdiscontinuance, which is then followed by the administration of at leastone compound disclosed supra.

The compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, is an oxytocin receptor antagonist.

As used herein, the term “oxytocin receptor antagonist” refers to acompound that functions by inhibiting (partially or completely) orblocking the oxytocin receptor (OT-R), thereby preventing activation ofthe receptor by oxytocin.

Generally, the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, is a vasopressin Via receptorantagonist.

As used herein, the term “vasopressin V1a receptor antagonist” refers toa compound that functions by inhibiting (partially or completely) orblocking the vasopressin V1a receptor (also known as Argininevasopressin receptor 1A), thereby preventing activation of the receptorby vasopressin. Vasopressin V1a receptor is one of the three majorreceptor types for the peptide hormone arginine vasopressin, the othersbeing V1b and V2 receptors.

Hence, the present invention relates to a solid oral formulationcomprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, and at least one or morepharmaceutically acceptable excipients, wherein said compound is anoxytocin receptor antagonist and/or a vasopressin V1a receptorantagonist.

Disorders associated with the oxytocin receptor activity and/orvasopressin Via receptor activity are selected from the non-limitinggroup comprising preterm labor, premature birth, embryo implantationfailure due to uterine contractions, dysmenorrhea, prematureejaculation, sexual dysfunction, endometriosis, infertility, benignprostatic hyperplasia, neuro-psychiatric disorders, autism, socialbehavior disorders, psycho-social stress, and/or cardiovasculardisorders.

The term “preterm labor” referring also to “premature labor”, shall meanexpulsion from the uterus of a viable infant before the normal end ofgestation, or more particularly, onset of labor with effacement anddilation of the cervix before the 37th week of gestation. It may or maynot be associated with vaginal bleeding or rupture of the membranes.

The term “dysmenorrhea” refers to a condition characterized by cyclicpain associated with menses during ovulatory cycles. The pain is thoughtto result from uterine contractions and ischemia.

The term “sexual dysfunction” refers to any disturbance or variation inthe four phases—excitement phase, plateau phase, orgasmic phase andresolution phase characterizing the human sexual response.

The term “neuro-psychiatric disorders” as used herein refers to mentaldisorders attributable to diseases of the nervous system, e.g.depression, obsessive-compulsive disorder and others.

The term “social behavior disorders” as used herein refers to emotionaldisturbance, inappropriate types of behavior or feelings, pervasive moodof unhappiness or depression and a range of perceived difficulties tobuild or maintain satisfactory interpersonal relationships

The term “psycho-social stress” as used herein refers to a conditionresulting from a perceived threat to the social status, social esteem,self-worth, respect or acceptance within a group, and that lead todevelopment of a stress response in the body and physical symptoms.

Assisted reproduction technologies are methods applied in humans for thetreatment of infertility and in animals for producing pregnancies.Infertility, which affects about 10% of human pairs worldwide, may betreated by in vitro fertilization and embryo transfer (IVF-ET) or inless complicated cases, by artificial insemination. Generally, a successof an embryo transfer is dependent on uterine receptivity, an entitythat is defined as an ability of uterus to provide optimal conditionsmandating proper implantation and embryo development. Basic componentsof uterine receptivity are uterine contractile activity and thecondition of endometrium.

Uterine contractions occurring during the embryo transfer may expelembryos from the uterus towards vagina or oviducts, which may be a causeof unsuccessful treatment, or in latter case a cause of extra uterinepregnancy, a serious, potentially life-threatening complication.

Hence, the present invention provides a solid oral formulation, for usein the treatment and/or prevention of disorders selected from the groupcomprising preterm labor, premature birth, dysmenorrhea, prematureejaculation, sexual dysfunction, endometriosis, embryo implantationfailure due to uterine contractions, infertility, benign prostatichyperplasia, neuro-psychiatric disorders, autism, social behaviourdisorders, psycho-social stress, and/or cardiovascular disorders.Preferably, said solid oral formulation is a dispersible tablet.

Preferably, the present invention provides a solid oral formulation foruse in the treatment and/or prevention of preterm labor, prematurebirth, dysmenorrhea and embryo implantation failure due to uterinecontractions.

The present invention also provides a process for the preparation of asolid oral formulation comprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, characterized by a step of wetgranulation.

In wet granulation, granules are formed by the addition on powderparticles of a liquid such as water, ethanol and isopropanol, eitheralone or in combination.

Preferably, the present invention provides a process for the preparationof a tablet comprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, characterized by a step of wetgranulation. More preferably, said tablet is a dispersible tablet.

Alternatively, the present invention provides a process for thepreparation of a solid oral formulation comprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, and at least one or morepharmaceutically acceptable excipients, characterized by the steps of:

(i) mixing the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, and at least one or morepharmaceutically acceptable excipients;

(ii) wet-granulating;

(iii) sieving the granules

(iv) blending with a lubricant such as glycerol dibehenate; and

(v) compressing the mixture obtained in step (iv) to form a tablet.

Preferably, said tablet is a dispersible tablet.

The tablet cores may vary in shape and be, for example, round, oval,oblong, cylindrical or any other suitable shape.

The present invention also provides a kit comprising a solid oralformulation comprising a compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, and at least one or morepharmaceutically acceptable excipients, and information for use thereof.The information contains instructions to administer the oral formulationto a subject in need thereof.

Generally, in the present invention the subject in need thereof ispreferably a mammal, most preferably a human, more preferably a woman.

For practical reason, the solid oral formulation of the presentinvention can be packaged in a unit dose. As used herein, the term “unitdose” refers to a solid oral formulation that is dispensed in a packageready to administer to the patient. Each unit dose contains apredetermined quantity of active product calculated to produce thedesired therapeutic effect, in association with at least one or moresuitable pharmaceutical excipients.

EXAMPLES Example 1: Purification of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime1.1 Synthesis of (3Z/E, 5S)-5-(hydroxymethyl)-1-[(2′-methyl-,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime

In the present invention, the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximewas obtained as a crude isomeric mixture comprising(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeand(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime.

Synthetic pathways of compounds used in the invention are for examplethose described in WO2004005249 and WO2005082848.

The compound“(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime”used herein is also defined as“(4Z,2S)-2-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl-carbonyl)]pyrrolidine-4-one-O-methyloxime”depending on the nomenclature used.

The compound(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximecan also be prepared following stages 1 to 6 as described below:

Stage 1: Preparation of 4-(2-methylphenyl)benzoic Acid

A solution of potassium carbonate (0.908 Kg, 6.57 mol, 2.06 wt) in water(2.20 L, 5.0 vol) was charged to a slurry of 4-bromobenzoic acid (0.441Kg, 2.19 mol, 1.0 wt) in water (4.41 L, 15.0 vol) at 15 to 25° C. Theresulting slurry was stirred at 15 to 25° C. and degassed three timesusing a vacuum-nitrogen purge cycle.Tetrakis(triphenylphosphine)palladium(0) (0.022 Kg, 0.019 mol, 0.05 wt)was charged and the vacuum-nitrogen purge cycle repeated. A solution ofo-tolylboronic acid (0.313 Kg, 2.30 mol, 0.707 wt) in methanol (3.53 L,8.0 vol) was degassed three times, using a vacuum-nitrogen purge cycle,and then charged to the 4-bromobenzoic acid slurry at 15 to 25° C. Thereaction mixture was heated to and maintained at reflux (71 to 78° C.)until reaction completion (The reaction is considered complete at 95%conversion), as determined by ¹H NMR analysis (d6-DMSO), typically 1.5to 2.5 hours. The reaction mixture was concentrated to 15 vol undervacuum at 40 to 45° C. Toluene (4.41 L, 10.0 vol) and tetrahydrofuran(4.41 L, 10.0 vol) were added to the residue, the resulting mixturestirred vigorously and acidified to pH 1 with hydrochloric acid (6M,2.00 L, 4.5 vol). The contents were stirred vigorously for 30 to 60minutes and the layers separated. Toluene (2.20 L, 5.0 vol) andtetrahydrofuran (2.20 L, 5.0 vol) were added to the aqueous phase andthe mixture stirred for 5 to 10 minutes. The layers were separated, thecombined organic phases filtered and concentrated to 10.0 vol undervacuum at 35 to 40° C. Toluene (4.41 L, 10.0 vol) was added to theresidue and the resultant concentrated under vacuum at 35 to 40° C. Thetetrahydrofuran content of the resulting slurry was determined by ¹H NMRanalysis (d6-DMSO) (Pass level: ≤1.0% w/w tetrahydrofuran with respectto toluene). The slurry was cooled to and aged at 0 to 5° C. for 30 to60 minutes, the solid collected by filtration and the filter-cake washedwith toluene (2.20 L, 5.0 val). The solid was dried in a vacuum oven at35 to 40° C. to give 4-(2-methylphenyl)benzoic acid [0.438 Kg, 94.1% th,99.3% w/w, 1H NMR (d6-DMSO) concordant with structure] as a pale yellowsolid.

Stage 2: Preparation of 4-(2-methylphenyl)benzoic Acid Chloride

Thionyl chloride (0.300 L, 4.11 mol, 0.685 vol) was added to a slurry of4-(2-methylphenyl)benzoic acid (0.435 Kg, 2.05 mol, 1.0 wt) in toluene(4.35 L, 10.0 vol) at 10 to 25° C. and the mixture heated to andmaintained at 75 to 80° C.3 until complete by 1H NMR analysis(d6-benzene,), typically 4 to 5 hours. Reaction completion wasaccompanied by the formation of a hazy solution. The resultant wasconcentrated to 5.0 vol by removal of toluene under reduced pressure at35 to 45° C. Toluene (2.18 L, 5.0 vol) was added to the concentrate andthe mixture concentrated to 4.0 vol by removal of toluene under reducedpressure at 35 to 45° C. The resultant was filtered through glassmicrofibre paper and the filter-cake washed with toluene (0.44 L, 1.0vol). The toluene solution of 4-(2-methylphenyl)benzoic acid chloride[0.439 Kg, 92.8% th, 100.9% w/w, 1H NMR (d6-benzene) concordant withstructure] was used directly in Stage 3.

Stage 3: Preparation of(4R)-4-hydroxy-1-[(2′-methyl-1,1′-biphenyl-4yl)-carbonyl]-L-proline

A solution of potassium carbonate (0.526 Kg, 3.81 mol, 1 0.2 wt) inwater (0.57 L, 1.3 vol) was charged to a solution of 4-hydroxy-L-proline(0.274 Kg, 2.09 mol, 0.625 wt) in tetrahydrofuran (2.20 L, 5.0 vol) andwater (0.44 L, 1.0 vol) at 15 to 25° C. followed by a line rinse ofwater (0.44 L, 1.0 vol). The mixture was cooled to 0 to 5° C. with rapidstirring and a solution of 4-(2-methylphenyl)benzoic acid chloride(0.438 Kg, 1.90 mol, 1.0 wt) in toluene (2.19 L, 5.0 vol) charged atthat temperature followed by a line rinse of toluene (0.44 L, 1.0 vol).The reaction mixture was warmed to 15 to 25° C. over 1 to 2 hours andstirred at this temperature until judged complete by TLC analysis. Water(2.20 L, 5.0 vol) was charged to the reaction mixture at 15 to 25° C.and the layers separated. The aqueous phase was acidified to pH 5 to 6with aq. hydrochloric acid (6M, 0.66 L, 1.5 vol) and then to pH1 withaq. hydrochloric acid (2M, 0.88 L, 2.0 vol) at 15 to 25° C. The mixturewas cooled to and aged at 0 to 5° C. for 30 to 60 minutes, theprecipitated solid collected by filtration, the filter-cake washed withwater (2×1.75 L, 2×4.0 vol) and toluene (0.88 L, 2.0 vol) and pulled dryon the filter for 12 to 24 hours. The collected solid was dried undervacuum at 40 to 45° C. until the water content by KF was ≤0.2% w/w toafford(4R)-4-hydroxy-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline[0.599 Kg, 97.0% th, 136.8% w/w, ¹H NMR (d₆-DMSO) concordant withstructure] as an off-white solid.

Stage 4: Preparation of1-(2′-methyl-1,1′-biphenyl-4-yl)carbonyl-4-oxo-L-proline

Triethylamine (1.80 L, 13.56 mol, 3.0 vol) was charged to a solution of(4R)-4-hydroxy-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline(0.598 Kg, 1.84 mol, 1.0 wt) in dimethyl sulfoxide (4.42 L, 7.4 vol) at15 to 20° C. Pyridine-sulphur trioxide complex (0.879 Kg, 5.52 mol, 1.47wt) was charged portion-wise at 15 and 25° C. and the reaction mixturestirred at that temperature until reaction completion, as determined byTLC analysis (typically 1 to 3 hours). 7 The reaction was quenched withaq. hydrochloric acid (3M, 4.80 L, 8.0 vol) at 0 to 30° C.,tetrahydrofuran (3.00 L, 5.0 vol) and heptanes (0.60 L, 1.0 vol)charged, the layers separated and the aqueous phase extracted withtetrahydrofuran (2×3.00 L, 2×5.0 vol). The combined organic phases werewashed with aq. hydrochloric acid (1 M, 2×1 0.20 L, 2×2.0 vol) andsaturated sodium chloride solution (2×1.20 L, 2×2.0 vol), the aqueouswashes combined and back-extracted with tetrahydrofuran (2×0.60 L, 2×1.0vol). The combined organics were dried over magnesium sulphate (1.794Kg, 3.0 wt), filtered, the filtercake washed with tetrahydrofuran (0.60L, 1.0 vol) and the filtrates concentrated under vacuum at 40 to 45° C.to give a pale brown foam. Ethyl acetate (6.00 L, 10.0 vol) was chargedto the foam, the contents stirred for 5 to 10 minutes to reachdissolution and the solvent removed under vacuum at 40 to 45° C. Thiswas repeated using ethyl acetate (6.00 L, 5.0 vol) until tetrahydrofuranwas not detected by ¹H NMR analysis (d6-DMSO). The residue was slurriedin ethyl acetate (4.80 L, 8.0 vol), activated carbon (0.084 Kg, 0.14 wt)added followed by a line rinse of ethyl acetate (3.00 L, 5.0 vol), theresultant heated to and maintained at 70 to 80° C. for 20 to 30 minutes,cooled to 40 to 55° C. and filtered through glass microfibre paper. Thefilter-cake was washed with ethyl acetate (1.50 L, 2.5 vol) and thecombined filtrates and wash concentrated to 2.5 to 3.5 vol under vacuumat 40 to 45° C. Crystallisation commenced during the concentration. Theconcentrate was transferred to a suitable vessel with a line rinse ofethyl acetate (0.30 L, 0.5 vol) and heated to 70 to 80° C. Additionalethyl acetate (0.30 L, 0.5 vol) was added as necessary to achievedissolution. Heptanes (1.80 L, 3.0 vol) was added at 70 to 80° C. andthe contents allowed to cool to between 15 and 25° C. over 1 to 2 hours.The slurry was further cooled to and aged at 0 to 5° C. for 2 to 3hours, filtered and the filtercake washed with ethyl acetate:heptanes(1:1, 0.60 L, 1.0 vol) at 0 to 5° C. followed by heptanes (3.0 L, 2.5vol). The collected solid was dried under vacuum at 40 to 45° C. to givel-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-4-oxo-L-proline [0.444 Kg,74.7% th, 74. 2% w/w, ¹H NMR (d6-DMSO) concordant with structure] as anoff-white solid.

Stage 5: Preparation of(4Z/E)-4-methoxyimino-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline

Triethylamine (0.40 L, 2.85 mol, 0.92 vol) was added to a solution of1-[(2′-methyl-1,1-biphenyl-4-yl)carbonyl]-4-oxo-L-proline (0.434 Kg,1.34 mol, 1.0 wt) in dichloromethane (4.40 L, 10.0 vol) at 10 to 25° C.followed by a line rinse of dichloromethane (0.43 L, 1.0 vol).Methoxylamine hydrochloride (0.130 Kg, 1.56 mol, 0.30 wt) was addedportionwise at 10 to 25° C. followed by a line rinse of dichloromethane(0.43 L, 1.0 vol) and the reaction mixture stirred at 10 to 25° C. untilreaction completion, as determined by TLC analysis (typically 3 to 5hours, TLC eluent: dichloromethane:methanol:acetic acid (90:10:1); uvvisualization). The solvent was removed under vacuum at 35 to 40° C.,the resultant dissolved in ethyl acetate (4.40 L, 10.0 vol) and washedwith aq. hydrochloric acid (1 M, 2×2.20 L, 2×5.0 vol). The acidic washeswere back extracted with ethyl acetate (2.20 L, 5.0 vol), the combinedorganic phases washed with sat. aq. sodium chloride solution (3.10 L,7.0 vol), dried over magnesium sulfate (0.300 Kg, 0.69 wt), filtered andthe filtercake washed with ethyl acetate (2.20 L, 5.0 vol). The filtrateand washes were combined and concentrated under vacuum at 35 to 40° C.to afford4-methoxyimino-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline[0.476 Kg, 100.6% th, 109.6% w/w, ¹H NMR (CDCl₃) concordant withstructure) as an off-white solid.

Stage 6: Preparation of (4Z/E,2S)-methyl-1-[(2′-methyl-1,1′-biphenyl-4-yl)-carbonyl]-4-methoxyiminopyrrolidine-2-carboxylate

Potassium carbonate (0.476 Kg, 3.44 mol, 1.0 wt) was added to a solutionof 4-methoxyimino-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]-L-proline(0.475 Kg, 1.35 mol, 1.0 wt) in acetone (4.75 L, 10.0 vol) and themixture cooled to 0 to 10° C. Dimethyl sulfate (0.128 L, 1.35 mol, 0.27vol) was added at 0 to 15° C. and the mixture stirred at 15 to 25° C.until reaction completion, as determined by TLC analysis, typically 3 to16 hours. The solvent was removed under vacuum at 40 to 45° C. and theresultant partitioned between ethyl acetate (3.80 L, 8.0 vol) and water(3.80 L, 8.0 vol). The layers were separated, the organic phase washedwith sat. aq. sodium chloride solution (2.85 L, 6.0 vol), dried oversodium sulfate (0.953 Kg, 2.0 wt) and filtered. The filter-cake waswashed with ethyl acetate (0.48 L, 1.0 vol) and the combined filtrateand wash concentrated under vacuum at 40 to 45° C. Excess ethyl acetatewas removed by azeotropic distillation with tetrahydrofuran (2×0.95 L,2×2.0 vol) under vacuum at 40 to 45° C. to give (4Z/E,2S)-methyl-1-[(2′-methyl-1,1′-biphenyl-4-yl)-carbonyl]-4-methoxyiminopyrrolidine-2-carboxylate [0.492 Kg, 99.6% th, 103.6% w/w, ¹H NMR(CDCl₃) concordant with structure] as a viscous brown oil.

Stage 7: Preparation of(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime

Lithium borohydride (0.049 Kg, 2.26 mol, 0.1 wt) was added portionwiseunder nitrogen to a stirred solution of (4Z/E,2S)-methyl-1-[(2′-methyl-1,1′-biphenyl-4-yl)-carbonyl]-4-methoxyiminopyrrolidine-2-carboxylate (0.492 Kg, 1.34 mol, 1.0 wt) intetrahydrofuran (2.31 L, 4.7 vol) and methanol (2.311 L, 4.7 vol) at 0to 30° C. The mixture was stirred at 15 to 25° C. to reactioncompletion, as determined by TLC analysis (Eluent: ethyl acetate;Visualisation: ninhydrin), typically 2 to 6 hours. The reaction mixturewas quenched with water (0.40 L, 0.8 val) at 15 to 25° C. and stirred at15 to 25° C. for 16 to 20 hours. The resultant was concentrated undervacuum at 40 to 45° C. and the residue partitioned between water (2.46L, 5.0 vol) and ethyl acetate (4.92 L, 10.0 vol). The layers wereseparated, the organic phase washed sequentially with aq. hydrochloricacid (1 M, 2.46 L, 5.0 vol), sat. aq. sodium hydrogen carbonate solution(2.46 L, 5.0 vol) and sat. aq. sodium chloride solution (2.46 L, 5.0vol). The organic phase was dried over magnesium sulfate (0.985 Kg, 2.0wt), filtered and the filter-cake washed with ethyl acetate (0.50 L, 1.0vol). The combined filtrate and wash were concentrated under vacuum togive a crude isomeric mixture comprising(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeand(3E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime[0.395 Kg, 86.9% th, 80.3% w/w, 1H NMR (CDCl₃) concordant withstructure; 82.0% area by HPLC, 71.4:28.6 Z/E ratio] as a viscous brownoil. The oil was dissolved in toluene (0.40 L, 1.0 vol, with respect toweight of product) and stored until required.

1.2 Dry flash chromatography of crude(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime

A dry flash chromatography purification of the crude isomeric mixtureobtained following the protocol described above was attempted usingdifferent elution conditions. A crude mixture of(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime concentrated to dryness was re-dissolved in 2 volumetoluene and loaded onto a pad of SiO2 (5 wt) prior to elution using 25volume fractions of eluent. (FIG. 4) Fractions 1-5: eluted with puretoluene

Fractions 6-10: eluted with Toluene 1 MeOH 1% vol/vol

Fractions 10 to 15: eluted with Toluene/MeOH 2% vol/vol

The Z and E forms are shown by shaded spots. Fractions 8 to 13 werecombined and concentrated to dryness. The results show a recovery of75%. There was no improvement in the E/Z ratio. A minor gain of about 4%area in purity of the isomeric mixture (E+Z) was observed before andafter dry-flash chromatography (Table 1).

TABLE 1 Comparative impurity profile before and after dry-flashchromatography % area Impurity at E + Z- Impurity at RRT 1.12 RRT 0.7isomers RRT 1.08 (Ar—Ar—CH2OH) Before dry 4.6 91.3 <0.5 4.1 flash Afterdry- 2.5 95.6 <0.5 0.7 flash RRT: Relative retention time

The dry-flash chromatography of the crude isomeric mixture does notallow the purification of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime. The E/Z ratio pre and post dry-flash remain in the rangeof 30/70 to 40/60.

Furthermore, such an approach should be considered on the basis of thescale at which the operation has to be carried out. On a 20 L scale,this operation would not be a time saving approach.

1.3 Assessment Toward Crystallization of the Pure Z from the CrudeIsomeric Mixture

The first part of the assessment toward crystallisation of the pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime from the crude mixture(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime, has been looking at solubility and possiblecrystallisation conditions of the pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime. The results of the solubility/crystallisation testscarried out on 15 mg scale are reported in Table 2 below

TABLE 2 Qualitative solubility data for(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one O-methyloxime SolventDissolves in: Comment heptanes — insoluble in 20 vol toluene 2 vol coldDIPE 40 vol hot THF 4 vol cold tBuOH 6 vol hot MIBK 4 vol hot IPA 4 volhot

The initial solubility screen showed that pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime isomer is soluble in a range of solvents. On the basis ofthe above results, crystallisation by addition of anti-solvent wasexamined and the results reported in Table 3. The anti-solvent was addedto a warm solution ca 40-50° C. and allowed to cool to room temperature.

In particular, the water (anti-solvent) was added to a warm (40-50° C.)solution of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime in IPA until cloudiness was reached and the mixture wasallowed to cool to room temperature.

TABLE 3 Crystallisation via addition of anti-solvent Solvent AntisolventComment toluene 20 vol heptanes 39 vol oils out THF 10 vol heptanes 40vol oils out tBuOH 10 vol water 20 vol oils out MIBK 10 vol heptanes 40vol oils out IPA 20 vol water 160 vol very fine solid, oils out onstanding IPA 8 vol water 18 vol very fine solid, oils out on standingDMSO 10 vol water 12 vol gel NMP 10 vol water 28 vol oils out MeOH 10vol water 10 vol oils out DMSO 20 vol water 16 vol oils out acetone 10vol water 10 vol oils out DCM 10 vol heptanes 50 vol oils out

The IPA/water crystallisation conditions were applied to a crudeisomeric mixture. The toluene solution was first concentrated to drynessprior to dissolution in IPA (8 vol) and addition of water (18 vol).Unfortunately, this resulted in material de-mixing as oil.

In another experiment, the antisolvent was added to a solution of crude(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime (90.4% area purity, contained 0.5% w/w toluene and 3.7%w/w THF) at room temperature until cloudiness was reached and themixture was left to stand at room temperature (Table 4).

TABLE 4 Crystallisation by addition of water at 18-22° C. SolventAntisolvent Comment MeOH 5 vol water 3 vol oils out DMSO 5 vol water 3vol oils out

At this point of the investigation, no suitable conditions ofcrystallisation of the pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime or allowing isolation of solid containing(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime have been identified.

Further crystallisation attempts were carried out using crude isomericmixture of(3Z/E,5S)-5-(hydroxymethyl)-1l-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime. In all cases, the volume of solvents was smaller thanwhat used previously and based only on a single solvent. The crudematerial (E/Z ratio 33:67 and purity (E+Z) 79.52% area) used for thiscrystallisation was concentrated to a foam (Table 5).

TABLE 5 crystallisation from single solvent at lower volume MaterialSolvent Ageing in freezer Ageing in fridge ‘Pure Z’ Ethyl Crystallisesre- Stays in solution, with Acetate dissolves as warms and withoutseeding 1.8 vol after 2 days. Crude Does not crystallize n/a with orwithout seeding. ‘Pure Z’ Diethylether On addition of ether n/a 2.3 volat 18-22° C. starts to dissolve then crashes out again. Recovery 70%Used for seeding Crude Oils Crystallises recovery 41 Re-dissolves as %E/Z ratio 40/60 purity warms 85.4% area. (mother liquors E/Z ratio 20/80purity 62.1% area). Seeds not used. ‘Pure Z’ TBME Oils Stays insolution, with 2.3 vol Re-dissolves as and without seeding warms after 2days. Crude Oils Stays in solution, with Re-dissolves as and withoutseeding warms after 2 days.

Crystallisation using ethyl acetate followed by aging in a freezerovernight gave crystallisation using the pure(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime material, but quickly re-dissolved as the sample warmed.No crystals were observed using crude material in ethyl acetate evenwhen seeds were added.

Crystallisation using diethylether followed by aging in a fridge gavecrystallisation using the crude(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime material. The solid was collected in 41% recovery.Unfortunately, the collected solid had a slighter poorer E/Z ratio thanthe input material and a slightly higher chemical purity.

TBME as solvent for both pure Z and crude gave oiling after aging infreezer, and stayed in solution after aging in the fridge with andwithout seeds.

Suitable crystallization conditions of the crude isomeric mixtureallowing improvement of the Z/E ratio and of the purity of the isomericmixture (E+Z) have not been found.

1.4 Substantially pure form of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime 1.4.1 Small Scale Purification

The isolation procedure in substantially pure form of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime was performed by chromatography using a Biotage system(Biotage AB, SE-751 03 Uppsala, Sweden) of the crude isomeric mixtureisolated after reduction of the oxime ester (Stage 7 of Example 1).

Five distinct batches (No. 020, 180, 062, 068, 076) of the crudeisomeric mixture were purified by Biotage chromatography. Furthermore,different conditions were used regarding batches No. 068 and 076.Purification was performed with a 5% w/w spike of oxime methyl esteradded (No. 068), and with an overloaded Biotage column (No. 076).

Each chromatography was run using Biotage 40M cartridges (40 g silica)which had been pre-flushed with toluene. Toluene:MeOH (99:1 v/v) wasthen eluted and collected in 100 ml fractions (total volume 4 L),followed by a flush of toluene:MeOH (96:4 v/v).

Fractions were analysed by TLC (eluent: ethylacetate) to determine whichfractions could be discarded and which fractions contained Z isomer.These Z fractions were then analyzed by HPLC. The pass criteria for afraction was >96% Z isomer and <1.2% E isomer.

Surprisingly, the purification through Biotage chromatography of variousbatches was very efficient as the substantially pure form of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is purified at 99.4% (Batches No. 020, No. 062, No. 068)and at 99.2% (Batches No. 180, No. 076). In particular, the Biotagechromatography in presence of oxime ester removes 5% w/w oxime esterwithout detriment to recovery or quality (Batch No. 068) and a 25%overcharge of the Biotage column does not cause a decrease in yield orquality (batch No. 076).

TABLE 6 efficiency of the Biotage chromatography Batch yield of No.Input % E/Z Output % E/Z Z isomer 020 3.0 g Pure Z-fractions: 33% 85.7%area purity 1.0 g % E/Z: 30.5/69.5 98.8% area purity % E/Z: 0.6/99.4 1802.0 g Pure Z-fractions 45% 92.0% area purity 0.9 g % E/Z: 32.8/67.299.6% area purity % E/Z: 0.8/99.2 062 3.0 g Pure Z-fractions 43% 83.5%area purity 1.3 g % E/Z: 32.7/67.3 99.8% area purity % E/Z: 0.6/99.4Mixture: 11% 1.2 g 91.0% area purity % E/Z: 69.6/30.4 068 3.0 g spikedwith ~5% Pure Z fractions: 40% ester 1.2 g ~78% area purity 99.8% areapurity % E/Z: 32.7/67.3 % E/Z: 0.6/99.4 Mixture: 14% 0.6 g 98.8% areapurity % E/Z: 27.9/72.1 Pure E fractions: N/A 1.1 g 70.7% area purity %E/Z: 98.7/1.3 (19.3% ester) 076 3.8 g Pure Z fractions 37% 83.5% areapurity 1.4 g % E/Z: 32.7/67.3 99.8% area purity % E/Z: 0.8/99.2 Mixture:17% 1.8 g 95.0% area purity % E/Z: 63.6/36.4

1.4.2 Large Scale Purification

Various batches of crude(3Z/E,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime(0.392 kg, 1.16 mol, 1.0 wt) were charged to a Biotage 150 L SIM unit asan approximate 50% w/w solution in toluene and purified using 1%methanol in toluene (150 L) followed by 2% methanol in toluene (50 L),fraction size 5.0 L. The collected fractions were analysed by TLC¹⁵ andHPLC analyses, as appropriate. The fractions that were deemed to containclean(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime(criteria: Z-isomer ≥96.00% area, E-isomer ≤1.20% area) were combinedand concentrated under vacuum at 40 to 45° C. Absolute ethanol (2×2 L)was added to the residue and the solution concentrated under vacuum at40 to 45° C. until the foamy solid could be manipulated. The desiredproduct,(3Z,5S)-1-[(biphenyl-4-yl-carbonyl)-5-hydroxy-methyl]pyrrolidine-3-one-O-methyloxime(0.089 Kg, 22.7% w/w, ¹H NMR (CDCl₃) concordant with structure, 99.3%area by HPLC, 98, 4:0.9 Z/E ratio was obtained as an off-white to lightbrown solid.

TABLE 7 Summary of purification of different batches of (3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime in substantially pure form. Batch Input Output Yield% Z form % E form No. (kg) (kg) (% w/w) (% area) (% area) 12 0.392 0.08922.8 98.65 0.85 116 0.392 0.114 29 98.34 0.89 120 0.441 0.081 18.4 97.901.81 122 0.380 0.094 24.3 98.52 1.14 124 0.387 0.096 25.3 98.89 0.73 1260.390 0.132 33.8 98.40 0.95 128 0.526 0.010 2 98.20 0.83 130 0.453 0.08619 98.46 1.23 132 0.440 0.082 19.3 98.86 0.85 134 0.39 0.144 36.9 98.730.96 138 0.273 0.098 35.9 98.92 0.66 140 0.463 0.059 13.1 98.52 1.13 1420.462 0.084 18.4 99.37 0.48 144 0.442 0.126 29 99.1 0.68 146 0.409 0.13533.5 99.21 0.46 148 0.460 0.107 23.8 99.13 0.65 150 0.409 0.071 18 98.920.66 152 0.392 0.054 14.3 98.82 0.76 156 0.445 0.039 8.8 98.64 0.87 1580.392 0.06 15.3 98.73 0.63 162 0.435 0.150 34.5 98.94 0.79 164 0.4340.192 44.2 99.21 0.58 166 0.415 0.074 17.8 98.79 0.73 174 0.518 0.10820.8 99.11 0.64 176 0.342 0.072 21 98.88 0.77 178 0.415 0.074 17.8 99.070.71 180 0.353 0.174 49.3 99.03 0.82 182 0.270 0.178 65.9 99.10 0.53

Appropriate batches of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime(2.713 kg, 1.0 wt) isolated from the Biotage chromatography werecombined and dissolved in absolute ethanol (5.16 L, 2.0 vol) at 15 to25° C., clarified by filtration through glass microfibre paper and anabsolute ethanol wash (0.50 L, 0.2 vol) applied to the filter. Thecombined filtrates were concentrated portion wise under vacuum at 40 to45° C. The resultant was transferred to drying trays and dried undervacuum at 30° C. for 24 hours. The oven temperature was then increasedincrementally from 30 to 40° C. over 80 hours. The level of residualsolvent was determined by ¹H NMR analysis (CDCl₃) and when found to be<0.0% w/w the solid was passed through a 500 m aperture sieve. The solidwas returned to the oven and dried at 40 to 42° C. until the solventlevel was ≤0.40% w/w to afford(3Z,5S)-1-[(biphenyl-4-yl-carbonyl)-5-hydroxy-methyl]-pyrrolidine-3-one-O-methyloxime(2.633 Kg, 97.1% w/w, 1H NMR (CDCl₃) concordant with structure, 98.65%area by HPLC.

The combination procedure is summarized below:

Input: 2.713 kg

Output: 2.633 kg

Yield: 97.1% w/w

Example 2: Capsule Oral Formulation 2.1 Bulk Preparation

Excipients were weighed directly into a beaker, which was transferredinto a thermostatic water bath until all excipients were molten at 60°C. Then, always under controlled temperature, small aliquots of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximewere added until all the drug was dissolved or dispersed, under magneticstirring, helix mixer or homogenizer.

2.2 Liquid Filling Capsules

The semi-solid bulk was maintained at 60° C. during the liquid fillingof the capsule shells. The filling step was performed at 60° C. (bothdosing pump and feeder) with an automatic lab-scale capsule-fillermachine. The machine was set to the correct dosage by weighing thefilled capsules.

Composition of Active Capsules (Table 8)

TABLE 8 Component mg/capsule (3Z,5S) 30.0 300.0 Gelucire 50/13TM 33.4334.0 LabrasolTM 16.6 166.0 Capsule, gelatine Size 00 Size 00Composition of Placebo Capsules (Table 9):

TABLE 9 Component mg/capsule Gelucire 50/13TM 63.4 634.0 LabrasolTM 16.6166.0 Capsule, gelatine Size 00 Size 00

Example 3: Granules Oral Formulation

Granules 10% were Prepared by Hot Melt Granulation (Table 10):

Hot melt granulation was conducted in a high shear granulator MiMiPro(Procept) using different set-ups depending on the batch size. Thegeneral method of manufacture is characterized by the steps of:

i) heat granulator water jacket to melting of waxy binder;

ii) screen powders into the heated bowls and mix gently;

iii) add waxy binder and mix gently; allow wax to soften;

iv) granulate for few minutes; rest the material for few minutes andgranulate again if necessary and

v) cool and screen the granules.

In particular, the jacketed vessel was used at 65° C. when Gelucire50/13 was used as binder. Cover temperature was set at 5° C. lower thanthe jacketed vessel's. Duration of phases depend on the desired particlesize distribution and on the batch size.

TABLE 10 Component Amount w/w (3Z,5S) 10.0% Saccharose 10.0% Acdisol5.0% Gelucire 50/13 15.0% Sodium Saccharine 0.2% Lactose anhydrous 59.8%

Hot melt granulation is an alternative technique of granulation: unlikethe traditional use of aqueous or organic solvents as binders, in thisprocess the agglomeration is obtained through the addition of a moltenbinder or a solid binder, which melts during the process and remains asa constituent of the formulation.

Composition of Granules 5.8% Prepared by Spray-Drying (Table 11):

The spray-drying process produces porous/hollow particles and amorphousforms of the sprayed material. This approach is used when dissolutionrate improvement is required. The spray-drying process consists of foursteps: atomisation of feed solution into a spray camera, spray-aircontact involving flow and mixing, drying of sprayed droplets atelevated temperatures and separation of dried product from air.

The granules 5.8% were prepared by spray-drying of (3Z,5S), in presenceof HP-β-CD (hydroxypropyl-β-cyclodextrin) in hydroethanolic solution.(3Z,5S) is an amorphous material that forms clumps of particles inwater, which reduces the drug dissolution rate. Therefore, a hydrophilicexcipient, spray-dried together with (3Z,5S) was used to improve thedissolution rate of the drug by preventing the aggregation in water.HP-β-CD was selected as hydrophilic excipients.

HP-β-CD spray dried product was obtained from the hydroalcoholicsolution both using the Mini AirPro or Buchi equipment. The feedingsolution was prepared by mixing an equal volume of a HP-β-CD (100 g in200 ml) aqueous solution and an (3Z,5S) (24 g in 200 ml) ethanolicsolution that was left for 24 hours under agitation at room temperature.The spray-drying conditions in the fluid bed Mini AirPro were: blowerspeed 1 m³/min, nozzle pressure 1 bar, liquid speed 3, inlet airtemperature 70° C.

Spray-dried materials presented very poor flow properties precludingtheir use for sachet filling. To obtain an easy handling powder, drygranulation and ethanolic wet granulation were the processes applied tothe (3Z,5S)—HP-β-CD spray-dried material. The dissolution rate of(3Z,5S) after spray-drying with HP-β-CD was almost instantaneous, in 15minutes almost all drug was dissolved.

The granulation process by ethanolic wet granulation was performed onthe (3Z,5S)—HPβCD spray-dried material. It did not modify thedissolution rate of the drug when compared to the (3Z,5S)—HPβCDspray-dried material.

It produced a material that showed almost instantaneous dissolution ofthe drug (Table 11).

TABLE 11 Component Amount w/w (3Z,5S) 5.8% Saccharose 10.0% Acdisol 3.0%Sodium saccharine 0.2% HP-β-CD 24.2% Poloxamer 188 2.0% Avicel RC 59110.0% Avicel PH 112 10.0% Lactose monohydrate 34.8%

Example 4: Conventional Tablet Oral Formulation

A solution containing (3Z,5S) dissolved in Labrasol:Ethanol 3:1 v/v wasprepared by heating at 45° C. and adding stepwise the required amount ofdrug. Zeopharm 600 was dried for 2 hours into a vacuum oven at 50° C.The addition of the solution on Zeopharm 600 bed was carried out into a1900 ml bowl at 5 ml/min. The granulator was set as follows: impeller at900 rpm, chopper at 3500 rpm, and cover temperature at 80° C. To removemost of the solvent, the material was left overnight at roomtemperature, and 3.5 hours in a vacuum oven at 50° C. As thegranule-adsorbate particle size was slightly high, the material wassieve-milled first through a 1.5 mm sieve and then through a 1 mm sieve.The production yield, including the milling step, was 89.68%. Thegranule-adsorbate (87.5%) was then mixed with AcDiSol (4%), Compritol888 ATO (3.5%), GL100 (0.2%) and Zeopharm 600 (4.8%) for 20 minutes at22 r.p.m in the Turbula mixer. The final blend for tabletting possesseda good (3Z,5S) content uniformity. The conventional tablets wereproduced by compression of the granules using the eccentric tablettingmachine EK-0.

Conventional tablets of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, were prepared using carriers ofcalcium silicate (Table 12).

TABLE 12 Component Amount w/w (3Z,5S)  35% Acdisol   4% Calcium silicate(Zeopharm 600) 32.7% Compritol ATO 888  3.5% Labrasol 24.6% Rx GL 100 0.2%

Example 5: Dispersible Tablet Oral Formulation

A 850 g batch granulate was produced and then compressed into tablets. A5000 ml vessel set-up was used for this preparation. Calcium silicatewas vacuum dried prior to use. The wet granulation was conducted with(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeand all excipients (with the exception of the lubricant) in a high sheargranulator at room temperature with only the cover of the granulatorheated (65° C.).

In the granulation process after a gentle pre-mix phase, an alternationof liquid addition and mixing phases of few minutes duration was set-up.

A total amount of 80 ml of ethanol was necessary to obtain suitablegranules. A final milling and/or sieving step was necessary to obtain abetter granule size distribution. The resulting granules were blendedwith the lubricant before compression.

A single punch eccentric tabletting machine was used.

The compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,and/or an active metabolite thereof, is in the form of dispersibletablets containing 50 or 200 mg of active drug substance (Table 13).

TABLE 13 Amount Component mg mg % (w/w) Function (3Z,5S) 50.0 200.0020.0 Active drug substance Calcium silicate 12.5 50.0 5.0 Carrier PVP30K 2.5 10.0 1.0 Binder Poloxamer 188 5.0 20.0 2.0 Wetting agent Sodium12.5 50.0 5.0 Disintegrant croscarmellose Microcrystalline 37.5 150.015.0 Diluent cellulose 112 Lactose monohydrate 119.5 478.0 47.8 DiluentSodium saccharine 0.5 2.0 0.2 Sweetener Glycerol dibehenate 10.0 40.04.0 Lubricant Total 250.0 1000.0 —

Example 6: Dimensions of the Tablets

For example, the shape and dimensions of the tablets are the following:

TABLE 14 Tablet or dispersible tablet Shape and dimensions  50 mgCapsule shape; 14 × 6 mm or 13 × 6 mm 200 mg Capsule shape; 22 × 9 mm or19 × 9 mm

Example 7: Pharmacokinetics Studies in the Dog

A pharmacokinetic study was conducted to measure the plasmaconcentration of the compound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximefollowing oral administration of said compound to female Beagle dogs.Following the protocol (Table 15), 5 dogs were administered with an oralformulation composed of liquid filled capsules (“Reference capsule”), 5dogs were administered with an oral formulation composed of granules 10%(formulation 1), 5 dogs were administered with an oral formulationcomposed of granules 5.8% (formulation 2), 5 dogs were administratedwith a dispersible tablet (formulation 3), 5 dogs were administered witha conventional tablet (formulation 4). Also 5 dogs were administered bythe IV route of administration (“Reference IV”) a solution of thecompound of formula(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloximeat 15 mg/kg. Blood sampling was performed at 0, 0.25, 0.5, 1, 2, 4, 6,8, 24, 48 and 72 h.

TABLE 15 Group Reference Reference IV capsule Formulation 1 Formulation2 Formulation 3 Formulation 4 Formulation Solution Liquid GranulesGranules Dispersible Conventional of (3Z,5S) filled 10% 5.8% tablettablet capsules 200 mg 200 mg Dose mg/kg 15 mg/kg 300 mg/ 20 20 23.4(3.4) 23.3 (3.2) dog (200 mg/dog) (200 mg/dog) Volume of 1 capsule 5ml/kg 5 ml/kg 50 ml/dog 50 ml/dog administration size 00

7.1 Plasma Concentration Profiles of Solid Oral Formulations in the Dog

FIG. 1A shows the plasma concentration vs. time profile of the differentformulations over the time period from 0 to 72 h. FIG. 1B shows anenlargement of FIG. 1A for the time period from 0 to 12 h. The curvecorresponding to formulation 3 (dispersible tablet) shows that themaximum concentration of the active substance is detected rapidly atabout 0.5-1 hour following its administration. In comparison, themaximum concentration of the active substance is detected at about 2-4hours following administration of formulation 4 (conventional tablet).

Individual plasma profile for each dog are presented on FIG. 2(formulation 3), and FIG. 3 (formulation 4) to show the inter-animalvariability.

7.2 Pharmacokinetic Parameters of Solid Oral Formulations in the Dog

In pharmacokinetics, the bioavailability is measured by calculating thearea under the curve (AUC) of the product concentration vs. timeprofile. The absolute bioavailability compares the bioavailability ofthe product in systemic circulation following oral administration withthe bioavailability of the product following intravenous administration.

“D” as used herein refers to the dose that is the amount of drugadministered.

“Cmax” as used herein refers to the peak plasma concentration of a drugafter administration wherein the concentration is the amount of drug ina given volume of plasma.

“Tmax” as used herein refers to the time to reach Cmax.

“T_(1/2)” as used herein refers to the elimination half-life as the timerequired for the concentration of the drug to reach half of its originalvalue.

“AUC” as used herein refers to the area under the curve that is theintegral of the concentration-time curve (after a single dose or insteady state).

“F %” as used herein refers to the bioavailability that is thesystemically available fraction of a drug. The index of bioavailabilityafter oral administration is calculated by the following equation usingthe AUC found after i.v. administration:F%=(AUC_(OS)/AUC_(IV))×(Dose_(IV)/Dose_(OS))×100

TABLE 16 Group Reference Reference IV Capsule 1 2 3 4 Formulation IVCapsule Granules Granules Dispersible Conventional 10% 5.8% tablettablet Route of IV OS OS OS OS OS administration Dose 15 34.4 (±4.0) 2020 25.7 (±2.3) 23.3 (±3.2) (mg/kg) C (0.25 h) — 13 (±15) 3590 (±1450)4523 (±2757) 5108 (±1955) 88 (±54) (ng/ml) Cmax 17071 (±6162) 15243(±3194) 7488 (±2236) 8722 (±2036) 13966 (±3217) 9245 (±2869) (ng/ml)Tmax (h) — 4 (4-4) 1 (0.5-2) 0.5 (0.5-0.5) 1 (0.5-1) 2 (2-4) AUC 38684(±2310) 80139 (±18793) 32775 (±9120) 29893 (±7483) 58952 (±5770) 53563(±7570) (h * ng/ml) T_(1/2) (h)  9.5 (±4.4) 7.1 (±1.2) 5.9 (±3.9) 4.7(±0.9) 6.6 (±0.6) 5.0 (±1.7) F (%) — 90 (±15) 64 (±18) 58 (±15) 89 (±9)89 (±13) Numbers in brackets represent the standard deviation.

Formulations 1 and 2 (granules) showed equivalent responses and werecharacterized by a favorable fast compound absorption (Table 16). Theirabsolute bioavailability was good (about 60%), even though lower thanthe other formulations tested, but associated with a moderatevariability between animals. Dispersible tablets Formulation 3 showed arate of absorption comparable to granules followed by a decay comparableto the Reference Capsule formulation. A very high compound exposurehighlighted by the absolute bioavailability (higher than afteradministration of granules and comparable to the liquid filled capsulereference formulation) was observed. Conventional tablets Formulation 4gave a delayed absorption compared to Formulation 3 with a roughlysimilar bioavailability.

Formulation 3 appears overall the most suitable for the indication ofpreterm labor. By comparison with the Reference Capsule formulation (300mg/dog, liquid filled capsules), the results highlight a faster compoundabsorption (about 37% of the amount found at Cmax already found at thefirst sampling time, 0.25 h), Cmax reached at earlier time (medianTmax=1 h), terminal elimination rate comparable (T_(1/0) of about 7 h)and overall more uniform responses between animals. Formulation 3exposure was equivalent to the Reference Capsule formulation and alsoabsolute bioavailability was equivalent to that of the Reference Capsuleformulation (89% vs. 90%).

7.3 Individual Plasma Concentration Profiles of Formulation 3 in the Dog

Route of administration: Oral

Administered dose of (3Z,5S): 200 mg/dog

Formulation 3: 200 mg dispersible tablet

Dose regimen: Single

TABLE 17 Animal No. (3Z,5S) plasma concentration in mg/mL Sampling time(h) 6F 7F 8F 9F 10F 0 (pre-dose) 1.5 2.3 * 3.4 * 0.25 8122 3965 29374994 5522 0.5 14107 9206 6913 16555 9070 1 18285 11934 11575 9137 114792 8175 7289 8341 7476 7942 4 5151 5188 6589 5660 4924 6 2508 2807 34342849 3027 8 1343 1626 2456 1819 3389 24 70 166 422 355 36 48 5 8.7 12 2212 72 3.7 2 2.1 2.3 3.4 * = Below the lower limit of quantification (1mg/mL)

Remarquably, at 0.5 hour time point following administration of thedispersible tablet, the concentration in blood of the compound offormula (3Z,5S) is comprised between 59% to 100% of Cmax indicating thatsaid formulation is suitable for providing a rapid onset of action(Table 17).

7.4 Individual Pharmacokinetic Parameters of Formulation 3 in the Dog

TABLE 18 Animal No. PK parameter 6F 7F 8F 9F 10F Cmax (ng/ml) 1828511934 11575 16555 11479 Tmax (h) 1 1 1 0.5 1 C (0.25 h) 8122 3965 29374994 5522 (ng/ml) AUC 56242 52929 67865 61111 56615 (h * ng/ml) T_(1/2)(h) 6 5.8 5.7 6.6 7.2 F (%) 85 80 102 92 85

The maximum concentration in blood of the compound of formula (3Z,5S) isreached at a time Tmax between 0.5 to 1 hour following administration ofFormulation 3 (dispersible tablet). In addition, Formulation 3 ischaracterized by a bioavailability of the compound of formula (3Z,5S)comprised between 80-100% (Table 18).

7.5 Individual Plasma Concentration Profiles of Formulation 4 in the Dog

TABLE 19 Animal No. (3Z,5S) plasma concentration in Sampling time ng/mL(h) 1F 2F 3F 4F 5F 0 (pre-dose) * * * * * 0.25 152 17 53 116 104 0.52217 2379 1680 2926 1945 1 3907 3031 3241 4339 4482 2 7759 5761 66719227 13492 4 5171 5646 9983 7085 7848 6 2806 4822 4307 3856 3185 8 15274195 3051 2713 2211 24 87 78 53 92 172 48 1.8 3.1 1.9 4.5 4.5 75 * * *1.2 1.6 * = Below the lower limit of quatification (1 ng/mL)

The maximum concentration in blood of the compound of formula (3Z,5S) isreached at a time Tmax between 2 to 4 hours following administration ofFormulation 4 (conventional tablet).

At 0.5 hour time point following administration of Formulation 4(conventional tablet), the concentration in blood of the compound offormula (3Z,5S) is comprised between 14% to 41% of Cmax (Table 19) avalue markedly lower than for formulation 3 which is characterized by ahigher concentration of the compound of formula (3Z,5S) comprisedbetween 59% to 100% of Cmax (Table 17).

7.6 Individual Pharmacokinetic Parameters of Formulation 2 in the Dog

TABLE 20 Animal No. PK parameter 11F 12F 13F 14F 15F Cmax (ng/ml) 70299050 9897 6347 11287 Tmax (h) 0.5 0.5 0.5 0.5 0.5 C (0.25 h) 3521 12754512 4445 8865 (ng/ml) AUC 21062 34881 39126 23931 30466 (h · ng/ml)T_(1/2) (h) 3.3 5.3 4.5 5.1 5.4 F (%) 41 68 76 46 59

Formulation 2 (granule 5.8%) is characterized by a bioavailability ofthe compound of formula (3Z,5S) comprised between 41-76% (Table 20)markedly lower than for formulation 3 which is characterized by a veryhigh bioavailability comprised between 80-100% (Table 18).

Thus, the dispersible tablet formulation 3 displays characteristics thatare suitable for providing a fast onset of action and highbioavailability for the treatment of preterm labor. In contrastconventional tablet (formulation 4) or granules (formulation 2) do notmeet the pharmacokinetic requirements for treating preterm labor.

Example 8: Pharmacokinetics Studies in Human

Study Protocol

Twelve healthy Caucasian women aged 54-62 years (mean 58.3 years), witha weight of 51 to 67 kg (mean 60.6 kg±5.1) and a body mass index rangingbetween 19.4-25.5 kg/m2 (mean 23.12 kg/m2±2.05) were enrolled. They wereadministered, on three separate treatment periods of one week, eithertwo consecutive (3Z,5S) doses of 600 mg/day (administered to subjectsusing 3 dispersible tablets of 200 mg in 150 ml of water) or twointramuscular injections of 12 mg/day betamethasone or both drugs incombination.

Betamethasone (Célestène, Schering-Plough, France) was administered byintramuscular injection of 12 mg/3 ml into the gluteus muscle, which isa recommended dose for antenatal betamethasone in preterm labor for theprevention of respiratory distress syndrome in neonates.

One subject (Subject S6) was withdrawn from the study after the firsttreatment period, due to high pre-dose blood pressure in the secondtreatment period. Therefore, pharmacokinetic parameters of the treatmentwith (3Z,5S) dispersible tablets were calculated for 12 subjects,whereas pharmacokinetic parameters of the combination treatment wereassessed for 11 participants.

During each treatment period of one week, blood samples were collectedfor the analysis of (3Z,5S) and betamethasone at time points 0(pre-dose) and at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 20and 24 hours after first dose on day 1. The plasma was prepared andstored below −20° C. All samples were analysed using validated LC-MS/MSmethods. For the analysis of (3Z,5S), the lower limit of quantificationwas 1.0 ng/ml. For betamethasone analyses, the lower limit ofquantification was 0.5 ng/ml.

Pharmacokinetic parameters were estimated by non-compartmental methodsusing the Phoenix® WinNonLin® version 6.3 (Pharsight). The followingpharmacokinetic parameters were calculated for (3Z,5S) during eachtreatment period: measured maximum concentration (Cmax), time to Cmax(Tmax), and area under the plasma concentration-time curve (AUC).

8.1 Individual Plasma Concentration Profiles of (3Z,5S) DispersibleTablet (Formulation 3) in Human

Tables 21 to 23 show plasma concentration profiles of (3Z,5S) andpharmacokinetic parameters concerning (3Z,5S) which was administered tosubjects using 3 dispersible tablets of 200 mg in 150 ml of water.

TABLE 21 Subject S Plasma concentration of (3Z,5S) in ng/ml Time S1 S2S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 0.25 1190 270 335 867 574 234 481 4071730 1610 409 1990 0.5 3020 1170 1310 3120 2020 860 1550 1510 2630 26402510 2690 0.75 3010 1450 1810 2760 2220 1400 2090 1440 2700 2120 23503070 1 3100 2410 1810 3160 1830 1560 1440 1450 2670 2180 1940 2900 1.53280 3380 1650 4070 1600 1770 1790 1370 2570 3190 2990 3600 2 3170 31501520 3820 1550 1940 2440 2100 3050 2980 4100 3630 3 3060 3360 1540 30701560 2060 2280 2060 2660 2980 3410 3570 4 2870 2600 2300 2940 1990 30602290 2160 2730 2770 3300 3460 6 2400 2200 1910 1910 1580 2000 1800 16702280 1780 1960 1980 8 1710 1870 1800 1940 1310 1670 1560 1290 1690 15501620 1760 10 1510 1700 1620 1560 1130 1490 1370 973 1570 1430 1490 149012 1420 1870 1720 1630 949 1380 1250 794 1510 1400 1650 1410 16 10001040 1030 1170 640 981 762 586 928 844 1020 866 20 804 868 718 978 554812 654 444 698 818 827 699 24 765 680 567 843 385 792 501 327 555 560759 612

TABLE 22 Subject S % of Cmax Time S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S120.25 36% 8% 15% 21% 26% 8% 20% 19% 57% 50% 10% 55% 0.5 92% 35% 57% 77%91% 28% 64% 70% 86% 83% 61% 74% 0.75 92% 43% 79% 68% 100% 46% 86% 67%89% 66% 57% 85% 1 95% 71% 79% 78% 82% 51% 59% 67% 88% 68% 47% 80% 1.5100% 100% 72% 100% 72% 58% 73% 63% 84% 100% 73% 99% 2 97% 93% 66% 94%70% 63% 100% 97% 100% 93% 100% 100% 3 93% 99% 67% 75% 70% 67% 93% 95%87% 93% 83% 98% 4 88% 77% 100% 72% 90% 100% 94% 100% 90% 87% 80% 95% 673% 65% 83% 47% 71% 65% 74% 77% 75% 56% 48% 55% 8 52% 55% 78% 48% 59%55% 64% 60% 55% 49% 40% 48% 10 46% 50% 70% 38% 51% 49% 56% 45% 51% 45%36% 41% 12 43% 55% 75% 40% 43% 45% 51% 37% 50% 44% 40% 39% 16 30% 31%45% 29% 29% 32% 31% 27% 30% 26% 25% 24% 20 25% 26% 31% 24% 25% 27% 27%21% 23% 26% 20% 19% 24 23% 20% 25% 21% 17% 26% 21% 15% 18% 18% 19% 17%

Following administration of the dispersible tablet of the invention, themaximum concentration Cmax in blood of the compound of formula (3Z,5S)is reached at a time Tmax between 0.5 to 4 hours. In particular, Tmax isin the range of 0.5 to 2 hours for 9 subjects, and in the range of 0.5to 1.5 hours for 5 subjects.

Of note, at 0.5 hour time point, the concentration in blood of thecompound of formula (3Z,5S) is comprised between 55% to 95% of Cmax for10 subjects, preferentially between 57% to 92% of Cmax, indicating thatthe solid oral formulation is rapidly absorbed and suitable forproviding a rapid onset of pharmacological action. For subjects S2 andS6, the plasma concentration of (3Z,5S) was below 55% at 0.5 h,respectively 35% and 28% of Cmax. However, it was respectively 71% and51% of Cmax at 1 h following administration of the dispersible tabletwhich concentration is suitable for the management of preterm labor.

8.2 Individual Pharmacokinetic Parameters of (3Z,5S) Dispersible Tablet(Formulation 3) in Human

TABLE 23 Subject S S1 S2 S3 S4 S5 S6 S7 Cmax ng/ml 3280 3380 2300 40702220 3060 2440 Tmax hour 1.5 1.5 4 1.5 0.75 4 2 AUC ng · h/ml 3855739042 32421 41209 24814 33388 29867 Subject S S8 S9 S10 S11 S12 Cmaxng/ml 2160 3050 3190 4100 3630 Tmax hour 4 2 1.5 2 2 AUC ng · h/ml 2408536317 34853 38944 38467

8.3 Plasma Concentration Profiles in Human for a Combination of (3Z,5S)and Betamethasone

Tables 24 to 26 show plasma concentration profiles of (3Z,5S) andpharmacokinetic parameters concerning (3Z,5S) administered incombination with Betamethasone.

TABLE 24 Plasma concentration of (3Z,5S) Subject S Plasma concentrationof (3Z,5S) in ng/ml Time S1 S2 S3 S4 S5 S7 S8 S9 S10 S11 S12 0.25 41.8597 322 486 264 536 359 396 1150 934 934 0.5 210 3240 978 1890 1250 25101100 2420 2590 2040 1980 0.75 407 5290 1130 2210 1780 2910 1610 25702490 2060 3010 1 593 5320 1100 2290 1580 2600 1730 2250 2350 1930 30201.5 1050 4940 1350 3550 2010 2620 1860 2300 3130 1920 3430 2 1050 47502180 4230 2080 3290 2400 2270 3520 4170 3550 3 1480 4480 3500 3620 22803540 2520 2340 3150 4440 3780 4 2040 4130 3500 3880 2350 3430 2540 23103340 4020 3550 6 1440 2510 1710 2290 2050 2710 1600 2910 2350 2690 26708 1470 1950 1530 2180 1680 2180 1250 2280 1820 2300 1970 10 1570 20901250 2120 1270 1960 981 2120 1620 1840 1750 12 1300 1870 1430 1930 10401750 1000 1970 1430 1630 2130 16 521 1140 1000 1330 693 935 550 959 869951 1010 20 512 1100 943 1240 481 831 471 825 746 786 877 24 418 828 7121020 353 569 383 605 533 650 657

TABLE 25 % of Cmax Subject S % of Cmax Time S1 S2 S3 S4 S5 S7 S8 S9 S10S11 S12 0.25 2% 11% 9% 11% 11% 15% 14% 14% 33% 21% 25% 0.5 10% 61% 28%45% 53% 71% 43% 83% 74% 46% 52% 0.75 20% 99% 32% 52% 76% 82% 63% 88% 71%46% 80% 1 29% 100% 31% 54% 67% 73% 68% 77% 67% 43% 80% 1.5 51% 93% 39%84% 86% 74% 73% 79% 89% 43% 91% 2 51% 89% 62% 100% 89% 93% 94% 78% 100%94% 94% 3 73% 84% 100% 86% 97% 100% 99% 80% 89% 100% 100% 4 100% 78%100% 92% 100% 97% 100% 79% 95% 91% 94% 6 71% 47% 49% 54% 87% 77% 63%100% 67% 61% 71% 8 72% 37% 44% 52% 71% 62% 49% 78% 52% 52% 52% 10 77%39% 36% 50% 54% 55% 39% 73% 46% 41% 46% 12 64% 35% 41% 46% 44% 49% 39%68% 41% 37% 56% 16 26% 21% 29% 31% 29% 26% 22% 33% 25% 21% 27% 20 25%21% 27% 29% 20% 23% 19% 28% 21% 18% 23% 24 20% 16% 20% 24% 15% 16% 15%21% 15% 15% 17%

Following administration of the combination (3Z,5S) dispersible tabletand betamethasone, the maximum concentration Cmax in blood of thecompound of formula (3Z,5S) is reached at a time Tmax between 1 to 6hours. In particular, Tmax is in the range of 2 to 4 hours for 9subjects, and in the range of 2 to 3 hours for 6 subjects.

At 0.5 hour time point, the concentration in blood of the compound offormula (3Z,5S) is comprised between 43% to 83% of Cmax for 10 subjectsindicating that the solid oral formulation administered in combinationwith betamethasone is rapidly absorbed and suitable for providing arapid onset of pharmacological action. For subjects S1 and S3, theplasma concentration of (3Z,5S) was below 43% at 0.5 h, respectively 10%and 28% of Cmax. However, it was respectively 51% and 62% of Cmax at 2 hfollowing administration of the dispersible tablet.

8.3 Pharmacokinetic Parameters in Human for a Combination of (3Z,5S)Formulation 3 and Betamethasone

TABLE 26 Subject S S1 S2 S3 S4 S5 S7 S8 S9 S10 S11 S12 Cmax 2040 53203500 4230 2350 3540 2540 2910 3520 4440 3780 ng/ml Tmax 4 1 3 2 4.03 3 46 2 3 3 hour AUC 24060 50306 34518 47627 28322 42044 25974 39688 3820843314 44082 ng · h/ml

Example 9: The Manufacturing Process of Dispersible Tablets

The manufacturing process of the dispersible tablets of the presentinvention comprises the following steps:

(i) Preparing a mixture consisting of 20% of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-one-O-methyloxime,5% of calcium silicate, 1% of PVP 30K, 2% of Poloxamer 188, 5% of sodiumcroscarmellose, 15% of Microcrystalline cellulose 112, 47.8% of lactosemonohydrate, 0.2% sodium saccharine, in weight based on the total weightof the tablet;(ii) Wet granulating in presence of ethanol and vacuum drying;(iii) Sieving the granules; (iv) Blending the granules with 4% ofglycerol dibehenate in weight based on the total weight of the tablet;(v) Tabletting.

The wet granulation is preferably conducted in a high shear granulatorat room temperature with a minimal amount of ethanol equivalent to atleast 7.4% (in weight based on the total weight of the tablet). Thevacuum drying is performed at room temperature

A sieving step is applied on the resulting granules. Sieved glyceroldibehenate is blended with the granules. The final blend is compressedwith an eccentric or rotary tablet press and adapted punches for thetargeted dispersible tablet strength.

Example 10: Stability Study of Dispersible Tablet (Formulation 3)

The determination of the (3Z,5S) content after dissolution ofdispersible tablets is performed by HPLC using the following parameters:

Apparatus: USPII paddle apparatus

Dissolution medium: 0.5% Sodium Lauryl Sulfate in water

Dissolution medium volume: 900 ml

Dissolution medium temperature: 37° C.+/−0.5° C.

Rotation speed: 50 rpm

Sampling time: 15, 30, 45, 60 and 120 min

Sampling volume: 3 ml

Separative technique: PALL Acrodisc PSF GxF/Glass 1.0 μm

The paddle assembly is arranged so that the bottom of the paddle was 2.5cm±0.2 cm from the inside bottom of the flask. The appropriate volume ofdissolution medium is poured into each one of the six dissolutionvessels. The medium is equilibrated at 37.0 C±0.5 C. A (3Z,5S)dispersible tablet is inserted into each vessel. The paddles rotationwas controlled at 50 rpm. At the designated time-points, 3 ml of mediumis taken from a zone midway between the surface of the dissolutionmedium and the top of the blade of the paddle. Then, the sample isfiltered through a PALL Acrodisc PSF GxF/Glass 1.0 μm directly into anHPLC vial for analysis.

Stability data are available at 1, 2 and 6 months storage time for(3Z,5S) dispersible tablets packaged in Alu/Alu blister packs.Parameters such as tablet appearance, (3Z,5S) content in % compared tothe initial content value, disintegration time and dissolution wereassessed (Tables 28 and 29).

The initial concentration value of (3Z,5S) measured by HPLC with adispersible tablet is 90.0-110.0%.

TABLE 28 Dispersible tablet of 50 mg (3Z,5S) Storage Water Content TimeTablet content (% initial Disintegr. % Dissolved Storage condition(months) appearance (% w/w) value) Time (sec) after 60 min Initial 0White capsule 4.5 102.9 42 101 shaped  5° C. 1 White capsule 4.7 102.419 100 shaped 2 White capsule 4.6 102.2 17 100 shaped 6 White capsule4.8 103.3 26 99 shaped 25° C./60% RH 1 White capsule 4.6 102.2 16 97shaped 2 White capsule 4.6 103.8 22 97 shaped 6 Off-white 4.7 105.7 2899 capsule shaped 40° C./75% RH 1 White capsule np np np np shaped 2Off-white 4.5  99.9 50 95 capsule shaped 6 Yellowish 4.6 102.1 113  94capsule shaped* np = not performed

TABLE 29 Dispersible tablet of 200 mg (3Z,5S) Storage Water Content TimeTablet content (% initial Disintegr. % Dissolved Storage condition(months) appearance (% w/w) value) Time (sec) after 60 min Initial 0White capsule 4.5 98.1 102 93 shaped  5° C. 1 White capsule 4.0 97.7 2699 shaped 2 White capsule 4.3 97.7 26 96 shaped 6 White capsule 4.1 99.728 99 shaped 25° C./60% RH 1 White capsule 4.0 98.0 26 96 shaped 2 Whitecapsule 4.2 98.9 28 100  shaped 6 Off-white 4.1 99.5 40 98 capsuleshaped 40° C./75% RH 1 White capsule np np np np shaped 2 Off-white 4.296.6 107 98 capsule shaped 6 Yellowish 4.1 98.3 170 96 capsule shaped*np = not performed

A rapid disintegration of the tablet is observed following dissolutionin the range of 20 to 40 sec at 25° C./60% RH.

The disintegration property of the dispersible tablet is likely due toits disintegrant component, for example, Sodium croscarmellose thatpromotes the breakup or disintegration of the tablet when placed in anaqueous environment and supports a fast dissolution profile. Inaddition, the wetting agent, for example, Poloxamer 188, facilitates thewater uptake during the disintegration and assists the drug dissolution.Furthermore, the carrier agent selected with a large surface area, forexample Calcium silicate, was also identified as a potential benefit forthe disintegration of the tablet.

TABLE 30 Dissolution profile of 200 mg dispersible tablet and content of(3Z,5S) at 15, 30, 45, 60, and 120 min. 15 min 30 min 45 min 60 min 120min Storage Time (3Z,5S) (3Z,5S) (3Z,5S) (3Z,5S) (3Z,5S) Condition(Months) Content % Content % Content % Content % Content % Initial 0 8994 96 97 99  5° C./AmbH 1 91 95 96 97 99 3 92 95 97 97 99 6 95 98 98 100101 25° C./60% RH 1 93 95 96 97 98 3 94 97 98 98 100 6 97 99 100 100 10240° C./75% RH 1 96 98 99 99 100 3 95 97 97 98 99 6 95 97 98 98 99

A rapid dissolution profile of the tablet is observed for the differentstorage conditions. In particular, at 25° C./60% RH, the content of(3Z,5S) measured at 15 min is between 90 to 100% of the initial value.

The invention claimed is:
 1. A method of treating infertility in a humanpatient, the method comprising administering to the patient adispersible tablet comprising a substantially pure form of(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime and one or more pharmaceutically acceptable excipients. 2.The method of claim 1, wherein the patient is undergoing an embryotransfer procedure, and whereby administration of the dispersible tabletreduces the likelihood of embryo implantation failure in the patient. 3.The method of claim 2, wherein the embryo transfer comprises in vitrofertilization (IVF) thereby producing an embryo, followed by transfer ofthe embryo to the patient.
 4. The method of claim 1, wherein the purityof the(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is from about 85% to about 99.9%.
 5. The method of claim4, wherein the purity of the(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is from about 90% to about 99.9%.
 6. The method of claim5, wherein the purity of the(3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyloxime is from about 95% to about 99.9%.